With the increasing flow of DNA sequence information from a variety of species, including humans, there is a need for new methods that can link function with gene sequence. We have developed an invention useful for probing the function of cell surface, transmembrane receptors, including the largest, and most pharmacologically important, gene classes in multicellular organisms, the seven transmembrane receptor (herein referred to as “7TMR”) class of genes. See generally Stadel, et al., Trends Pharmacol. Sci. 18(11): 430-7 (1997). Receptors often reside at the membrane surface of the cell and, upon binding of a ligand, activate an intracellular signaling cascade that mediates a particular cellular response. A crucial step in understanding the function of a particular receptor is to identify the ligand, or a surrogate ligand, that can bind and activate the receptor. The invention we describe here, which we dub the “C. elegans Chemosensory Bioassay,” allows nematodes to sense and respond to substances that may affect cell surface receptor activity. This invention can be used in a variety of ways, including, but not limited to: (1) screening and identifying substances that bind to and activate human 7TMRs; (2) screening for substances that antagonize human 7TMR activation; (3) identifying human 7TMRs that may respond to particular substances; and (4) evaluating the specificity and efficacy of substances on human 7TMR activation.
The instant invention represents a significant improvement over current technologies for probing transmembrane receptor function. Current methods for identifying activating for receptors are, at best, medium throughput (See Stadel, et al., supra.). Elaborate and sophisticated molecular assays designed to measure such parameters as calcium mobilization, cAMP, GTP-γ S binding, inositol phosphate production, MAP kinase activation, etc., are used to identify activating substances. These assays involve either sophisticated machines that can detect receptor activation in transgenic mammalian cell lines (Sullivan, et al., Methods Mol Biol 114:125-33 (1999)) or labor-intensive methods, such as microinjection of Xenopus oocytes followed by electrophysiological recording (Wagner, et al., Cell Physiol Biochem. 10(1-2): 1-12 (2000)) Such assays involve direct mechanical detection of receptor function.
By contrast, our invention links receptor activation to simple nematode behaviors, obviating the need for sophisticated instrumentation. In addition, the self-replicating biological nature of nematodes allows large numbers of animals to be produced in the laboratory, ensuring that our invention will be an inexpensive solution and will be amenable to high-throughput applications. For example, libraries of substances can be easily screened, as discussed below. Also, unlike mechanical detection of receptor activation, our invention does not require any prior assumptions about the parameters to be measured (calcium, cAMP, etc.). Instead, our invention uses simple biological behaviors in nematodes to assay receptor activation.
Cell surface receptors are a large collection of proteins that fit into a number of superfamilies by their structural similarities. These superfamilies include, but are not limited to: ion channels, transporters, and 7 transmembrane receptors (Saier M H Jr, J Membr Biol 175(3):165-180 (2000); Saier M H Jr, J Cell Biochem, Suppl 32-33:84-94 (1999); Bockaert J, Pin J P, EMBO 18(7): 1723-1729 (1999)). It is well established that many medically significant biological processes are mediated by such proteins. For example, the most common inherited disease among caucasians is cystic fibrosis, caused by mutations in the CFTR chloride channel (Frizzell R A Physiol Rev, 79(1 Suppl): S1-2 (1999)). In addition, a number of other disease states are linked to mutations in other ion channels (Weinreich F, Jentsch T J, Curr Opin Neurobiol 10(3):409-415 (2000)). One role of the transporter class of cell surface proteins is the uptake of neurotransmitters from synaptic clefts in the nervous system. These transporters are targets for therapeutic intervention in a variety of psychiatric disorders. For example, selective serotonin uptake inhibitors are used in the treatment of a number of disorders and include the widely prescribed drug Prozac (Masand P S, Gupta S, Harv Rev Psychiatry, 7(2):69-84 (1999)).
Among the cell surface receptor superfamilies, the 7 transmembrane receptor (7TMR) superfamily appears to be the largest in metazoan organisms (see Bockaert and Pin, supra). 7TMRs mediate a variety of cellular responses to extracellular stimuli. For example, 7TMRs bind or interact with a huge variety of ligands; including photons, small molecules, and large proteins (see Bockaert and Pin, supra). 7TMRs are be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson, et al., Endoc. Rev., 1989, 10:317-331). The 7TMRs include dopamine receptors, which bind to neuroleptic drugs used for treating psychotic and neurological disorders. Other examples of members of this family include, but are not limited to, calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1, rhodopsins, odorant, and cytomegalovirus receptors. Thus, these receptors mediate a large variety of biological and cellular responses to extracellular cues and include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.
For most cell surface receptors identified in genome sequencing efforts, the activating ligand is not known. However, given the wide variety and important nature of biological processes regulated by cell surface receptors, determination of the activating ligand is of fundamental pharmaceutical importance. For example, over the past 15 years nearly 350 therapeutic agents targeting 7TMRs have been successfully introduced onto the market (See Stadel, et al., supra). Therefore, finding out what ligands bind these 7TMRs will enable researchers to develop drugs to either agonize or antagonize the interaction of 7TMRs with their ligands. Our invention can be applied to this problem and is useful for probing the function of a large number of cell surface protein types by linking simple C. elegans behaviors to the activation of human 7TMRs.